Method for evaluating mesenchymal stem cell activity, method for culturing mesenchymal stem cells, method for producing therapeutic agent for liver dysfunction, and therapeutic agent for liver dysfunction

ABSTRACT

The present invention relates to a method for evaluating the activity level of mesenchymal stem cells, and a method for culturing mesenchymal stem cells using the evaluation method in the field of culturing mesenchymal stem cells for regenerative medicine, and further, a method for producing a therapeutic agent for liver dysfunction and a therapeutic agent for liver dysfunction. This method for evaluating mesenchymal stem cell activity according to the present invention comprises an assay step for assaying the amount of adenylate kinase 4 (AK4) in the mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4. 
     In this method, the activity level of mesenchymal stem cells can be evaluated from the assayed amount of AK4, for instance, if the mitochondrial activity level of mesenchymal stem cells is high, the mesenchymal stem cells can be evaluated as ideal for use as a therapeutic agent, while if mitochondrial activity of the mesenchymal stem cells is low and aging does not proceed, the mesenchymal stem cells can be evaluated as suitable for subculture.

TECHNICAL FIELD

The present invention relates to the field of culturing mesenchymal stem cells for regenerative medicine and, more specifically, to a method for evaluating the activity level of mesenchymal stem cells, and a method for culturing mesenchymal stem cells using the evaluation method, and further to a method for producing a therapeutic agent for liver dysfunction and a therapeutic agent for liver dysfunction.

BACKGROUND ART

In the field of regenerative medicine, use of mesenchymal stem cells for treating tissues in the human body has been attracting attention.

Mesenchymal stem cells are contained in bone marrow aspirates, umbilical cord blood, peripheral blood, and the like, and are cells having multipotency capable of differentiating into various types of cells, such as those in bones, cartilages, fat, heart, nerves, and liver. However, mesenchymal stem cells comprise, for example, about 0.05% of total cells in bone marrow aspirates, while it is necessary to collect a large amount for direct clinical use. Such direct clinical use is a heavy burden for the living body and, therefore, use of cells obtained by subculturing mesenchymal stem cells collected from living tissue is desirable.

Proliferation of mesenchymal stem cells to a desired amount by primary culture and subculture of a collected bone marrow aspirate is conventionally already known (Patent Literature 1).

PRIOR ART DOCUMENTS Patent Literature

-   [Patent Literature 1]

Japanese Patent Laid-Open No. 2011-67175.

SUMMARY OF INVENTION Problems to be Solved by the Invention

In Patent Literature 1, simple subculture for culturing mesenchymal stem cells to a predetermined amount is disclosed, but the activity (quality) of cultured mesenchymal stem cells is not considered.

As described in Patent Literature 1, the activity of mesenchymal stem cells is known to decrease (aging) as their culture continues. Moreover, subculture involves repetition of dividing mesenchymal stem cells cultured in a container into plural containers and culturing them; and, therefore, culturing conditions vary among the containers. Accordingly, final collections of cultured mesenchymal stem cells include aged and dead cells and there is a possibility of failing to acquire a sufficient quality and amount required for clinical use.

In view of such circumstances, the present invention provides a method for evaluating the activity level of cultured mesenchymal stem cells, as well as a method for culturing mesenchymal stem cells using the evaluation method, a method for producing a therapeutic agent for liver dysfunction, and a therapeutic agent for liver dysfunction.

Means for Solving the Problems

Accordingly, the invention of claim 1 relates to a method for evaluating the activity of mesenchymal stem cells, characterized by comprising a collection step for collecting a subject fluid containing mesenchymal stem cells such as a bone marrow aspirate, umbilical cord blood, or peripheral blood; an isolation step for isolating mesenchymal stem cells from the collected subject fluid; an assay step for assaying the amount of adenylate kinase 4 in the isolated mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4.

The invention of claim 3 relates to a method for culturing mesenchymal stem cells, characterized by comprising a collection step for collecting a subject fluid containing mesenchymal stem cells such as a bone marrow aspirate, umbilical cord blood, or peripheral blood; an isolation step for isolating mesenchymal stem cells from the collected subject fluid; a culturing step for culturing the isolated mesenchymal stem cells; a sampling step for sampling a part of cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4.

The invention of claim 10 relates to a method for producing a therapeutic agent for liver dysfunction, characterized by comprising a collection step for collecting a bone marrow aspirate; an isolation step for isolating a fluid containing mesenchymal stem cells from the collected bone marrow aspirate; a culturing step for culturing mesenchymal stem cells in the isolated fluid; a sampling step for sampling cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4.

The invention of claim 11 relates to a therapeutic agent for liver dysfunction, characterized in that the agent is produced by steps comprising: an isolation step for isolating a fluid; a culturing step for culturing mesenchymal stem cells in the isolated fluid; a sampling step for sampling cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4.

Advantageous Effects of Invention

In any of the inventions, the activity level of mesenchymal stem cells can be evaluated from the assayed amount of adenylate kinase 4; for instance, if the activity level of mesenchymal stem cells is high, the mesenchymal stem cells can be evaluated as ideal for use as a therapeutic agent.

Conversely, if the mitochondrial activity level of mesenchymal stem cells is high, then cells are aged in the view point of aging and the cells can be evaluated as not preferable to be used for subculture, while if mitochondrial activity of the mesenchymal stem cells is low and aging does not proceed, the mesenchymal stem cells can be evaluated as suitable for subculture.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a figure of experimental results illustrating the results of assaying the amount of adenylate kinase 4 when mesenchymal stem cells were cultured under conditions at 1% oxygen and when they were cultured under conditions at 20% oxygen.

FIG. 2 is an illustration that illustrates the relation between the amount of adenylate kinase 4 and the culturing step.

MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized by assaying an amount of adenylate kinase 4 in mesenchymal stem cells and evaluating the activity level of the mesenchymal stem cells by the assayed amount of adenylate kinase 4.

Adenylate kinase plays an important role in the maintenance of homeostasis of intracellular adenine nucleotides required for the living body to exhibit functions such as proliferation, differentiation, exercise, and metabolism normally and is an enzyme catalyzing the following reversible reaction.

2ADP (adenosine diphosphate)⇄ATP (adenosine triphosphate)+AMP (adenosine monophosphate)

Adenylate kinase has 4 isozymes: adenylate kinase 1 (AK1) locates in cytoplasm, adenylate kinase 2 (AK2) locates in the mitochondrial inner membrane and adenylate kinase 3 (AK3) and adenylate kinase 4 (AK4) locate in the mitochondrial matrix.

The functions of AK1, AK2, and AK3 have been gradually revealed in recent years but the function of AK4 is not well understood.

The present inventors conducted various experiments on AK4 and found as a result that the activity level of mesenchymal stem cells can be evaluated from the amount of AK4, thereby completing the present invention.

FIG. 1 illustrates the results of assaying the amount (expression) of AK4 by microarray analysis when human mesenchymal stem cells collected from a bone marrow aspirate were cultured under conditions at 1% oxygen and when they were cultured under conditions at 20% oxygen.

As apparent from the figure, while the amount of AK4 in mesenchymal stem cells cultured under conditions at 20% oxygen was 1 (in arbitrary unit, hereinafter abbreviated as a.u.), the amount of AK4 in mesenchymal stem cells cultured under conditions at 1% oxygen was 1.50 (in a.u.). It is well known that mesenchymal stem cells have higher mitochondrial activity under conditions at 20% oxygen than that under conditions at 1% oxygen. From this, it can be seen that when the amount of AK4 is high, the mitochondrial activity is low and the activity of mesenchymal stem cells (stem cell activity) is high and that when the amount of AK4 is low, the mitochondrial activity is high and the activity of mesenchymal stem cells (stem cell activity) is low.

The aforementioned amount of AK4 can be evaluated by western blotting. It can be also estimated by a commercially available flux analyzer (for example, XFe96 from Seahorse Bioscience Inc.).

In the present invention, the activity level of mesenchymal stem cells can be evaluated from the assayed amount of AK4 and, therefore, this evaluation can be used as follows.

First, mesenchymal stem cells having higher activity have higher effect as a therapeutic agent and, therefore, mesenchymal stem cells having high activity can be selected and used as a therapeutic agent, or can be used in a method for producing such a therapeutic agent.

Specifically, when a bone marrow aspirate is collected from a person with liver dysfunction, if the amount of AK4 assayed by steps comprising: a separation step for separating a fluid (the mononuclear cell component) from the bone marrow aspirate; a culturing step for culturing mesenchymal stem cells in the isolated fluid; a sampling step for sampling cultured mesenchymal stem cells; an assay step for assaying the amount of AK4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of AK4, is high, then the activity level of the mesenchymal stem cells is high and the mesenchymal stem cells can be used as a therapeutic agent for liver dysfunction. Moreover, the aforementioned steps can be used as a method for producing a therapeutic agent for liver dysfunction.

Second, when subculture is necessary for proliferating collected mesenchymal stem cells to a predetermined amount, mesenchymal stem cells having not too much expression of AK4 are preferably subcultured, since too much expression of AK4 in cultured mesenchymal stem cells results in inhibition of proliferative capacity when using as a therapeutic agent,

Specifically, when a subject fluid containing mesenchymal stem cells such as a bone marrow aspirate, umbilical cord blood, or peripheral blood is collected, if the amount of AK4 assayed by a collection step for collecting the subject fluid; an isolation step for isolating mesenchymal stem cells from the collected subject fluid; a culturing step for culturing the isolated mesenchymal stem cells; a sampling step for sampling a part of cultured mesenchymal stem cells; an assay step for assaying the amount of AK4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of AK4, is low, then the mitochondrial activity level in the mesenchymal stem cells is high and they are not used for subculture, while mesenchymal stem cells having a high amount of AK4 have a low mitochondrial activity level and, therefore, can be used for subculture.

In this case, when subculture involving dividing mesenchymal stem cells cultured in a container into plural containers and culturing is repeated a required number of times, plural mesenchymal stem cells having high and uniform amounts of AK4 may be selected to keep culture conditions in the containers as uniform and good qualities as possible. This makes it possible to obtain uniform and good qualities of mesenchymal stem cells in an amount sufficient for clinical use.

Third, when subculture is necessary for proliferating collected mesenchymal stem cells to a predetermined amount, if the number of times of the subculture can be predicted beforehand, then the amount of AK4 at the time of the start of each subculture can be selected so that the activity of the mesenchymal stem cells become high enough to obtain a good effect as a therapeutic agent when the last subculture is finished.

Accordingly, when subculturing, it is not necessary to select mesenchymal stem cells having the highest amount of AK4, but it is possible to select mesenchymal stem cells having the most suitable amount of AK4 in consideration of the number of times of subculture between the mesenchymal stem cells having the lowest amount of AK4, in which the mesenchymal stem cells are aged, and the mesenchymal stem cells having the highest amount of AK4.

The amount of AK4 when subculturing may be determined based on the period of cell culture and the number of times of subculture so that cells will have high or low activity at a certain time point.

EXAMPLES (Primary Culture Step)

-   (1) To mesenchymal stem cells prepared from myeloblasts, a culture     solution is added to a total volume of 30 mL, and 10 mL aliquots are     transferred into 3 flasks to start culturing in an incubator     (culturing step: see FIG. 2).

In this example, collected mesenchymal stem cells are divided into 3 groups in 3 flasks different in culture conditions and cultured.

-   (2) The total volume of medium is changed every 2-3 days (medium     change step).

(Subculture Step: First Time)

-   (1) 10,000 mesenchymal stem cells each are sampled from the 3 flasks     in the primary culture step (sampling step). -   (2) The amount of intracellular AK4 in each sample is assayed (assay     step). The amount of AK4 can be estimated by a flux analyzer as     described above. Alternatively, the amount can be precisely assayed     by western blotting or the expression analysis of mRNA. -   (3) Whether the assayed amount of AK4 is equal to or higher than a     predetermined threshold α or not is verified (determination step).     As this threshold α, for example, the value 1.1 a.u. in FIG. 1 can     be adopted. If the amount is equal to or higher than the value, then     the mitochondrial activity of the primary cultured mesenchymal stem     cells is low and aging does not proceed, and the mesenchymal stem     cells can be evaluated as suitable for subculture.

On the other hand, if the assayed amount of AK4 is lower than the predetermined threshold α, then it is estimated that the mitochondrial activity level in the mesenchymal stem cells is high and these cells are not used for subculture.

In FIG. 2, since the amounts of AK4 in the right and left mesenchymal stem cells among the mesenchymal stem cells divided into 3 flasks are equal to or higher than the threshold α, these cells are subcultured (see the mark @). Since the amount of AK4 in the mesenchymal stem cells in the central flask is lower than the threshold α, these cells are not subcultured (see the mark ×). Meanwhile, the mesenchymal stem cells in the central flask, which are not subcultured, are not abandoned at this time point, but culture is continued in an incubator.

On the other hand, the right and left mesenchymal stem cells, which are determined to be subcultured, are transferred into 3 flasks each and cultured in an incubator. In this example, the primary cultured mesenchymal stem cells are divided into 6 groups and the first subculture is performed.

(Subculture Step: Second Time)

-   (1) 10,000 mesenchymal stem cells each are sampled from the     aforementioned 6 flasks (sampling step). -   (2) The amount of AK4 in the sampled cells is assayed (assay step). -   (3) If the assayed amount of AK4 is equal to or higher than the     threshold α, the cells in the sampled flask are further transferred     into 3 flasks and the second subculture is performed in an     incubator.

In the case illustrated in FIG. 2, since the amounts of AK4 in the mesenchymal stem cells in the 4 groups with the mark @ among the mesenchymal stem cells divided into 6 groups in the first subculture are equal to or higher than the threshold α, the second subculture is performed for these 4 groups. Since the amounts of AK4 in the mesenchymal stem cells in the 2 remaining groups with the mark × are less than the threshold α, these cells are not subcultured. Meanwhile, the mesenchymal stem cells in the 2 groups, which are not subcultured, are not abandoned at this time point, but culture is continued in an incubator.

On the other hand, the mesenchymal stem cells in the 4 groups, which are determined to be subcultured, the mesenchymal stem cells in each group are transferred into 3 flasks each and cultured in an incubator. The mesenchymal stem cells are divided into 12 groups in total and the second subculture is performed.

(Cell Suspension Preparation Step)

-   (1) 10,000 mesenchymal stem cells each are sampled from the     aforementioned 12 flasks. Moreover, 10,000 mesenchymal stem cells     each are sampled as well from the other 3 flasks, which are not     subcultured. -   (2) The amount of AK4 in the sampled cells is assayed. -   (3) If the assayed amount of AK4 is equal to or lower than the     predetermined threshold β, for example, equal to or lower than the     threshold β=1.4 a.u., then these mesenchymal stem cells are     subjected to the separation of cells by washing the flask and total     200 mL of a cell suspension can be prepared by adding agents and a     physiological saline and used as a therapeutic agent.

On the other hand, if the assayed amount of AK4 is higher than the threshold β, then the mitochondrial activity level of the mesenchymal stem cells is low and the proliferative capacity decreases. Accordingly, such mesenchymal stem cells are unlikely to be effective when used as a therapeutic agent and they are abandoned. However, depending on the conditions, for example, when the total amount of the therapeutic agent is short, even mesenchymal stem cells having a low mitochondrial activity level are desirable to be used as a therapeutic agent rather than to be abandoned.

Although it is desirable to proliferate cells by subculture to reduce the patient burden, if a sufficient amount of mesenchymal stem cells can be collected, then the amount of AK4 in the mesenchymal stem cells is assayed after the primary cell culture is performed and cells having an assayed amount of AK4 that is equal to or lower than the threshold β can be used as a therapeutic agent without performing subculture.

Moreover, the numerical values of the aforementioned thresholds α and β for the amount of AK4 are examples and may be of course changed or adjusted to the most suitable value as appropriate depending on the required conditions such as the site to be used as a therapeutic agent and the necessary number of times of subculture. 

1. A method for evaluating the activity of mesenchymal stem cells, characterized by comprising: a collection step for collecting a subject fluid containing mesenchymal stem cells such as a bone marrow aspirate, umbilical cord blood, or peripheral blood; an isolation step for isolating mesenchymal stem cells from the collected subject fluid; an assay step for assaying the amount of adenylate kinase 4 in the isolated mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase
 4. 2. The method for evaluating the activity of mesenchymal stem cells according to claim 1, characterized in that the mesenchymal stem cells are mesenchymal stem cells collected from a bone marrow aspirate and cultured.
 3. A method for culturing mesenchymal stem cells characterized by comprising: a collection step for collecting a subject fluid containing mesenchymal stem cells such as a bone marrow aspirate, umbilical cord blood, or peripheral blood; an isolation step for isolating mesenchymal stem cells from the collected subject fluid; a culturing step for culturing the isolated mesenchymal stem cells; a sampling step for sampling a part of cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase
 4. 4. The method for culturing mesenchymal stem cells according to claim 3, characterized in that the culturing step comprises dividing the mesenchymal stem cells into plural groups and culturing the plural groups, and the sampling step comprises sampling a part of each of the plural groups of mesenchymal stem cells.
 5. The method for culturing mesenchymal stem cells according to claim 3, characterized in that the method comprises a subculturing step for subculturing the sampled mesenchymal stem cells if the assayed amount of adenylate kinase 4 is equal to or higher than a predetermined threshold α in the determination step.
 6. The method for culturing mesenchymal stem cells according to claim 3, characterized in that the sampled group of mesenchymal stem cells is not subcultured if the assayed amount of adenylate kinase 4 is lower than a predetermined threshold α in the determination step.
 7. The method for culturing mesenchymal stem cells according to claim 3, characterized in that the sampled mesenchymal stem cells are used as a therapeutic agent if the assayed amount of adenyate kinase 4 is equal to or lower than a predetermined threshold β in the determination step.
 8. The method for culturing mesenchymal stem cells according to claim 5, characterized in that the method comprises: a sampling step for sampling a part of the mesenchymal stem cells subcultured in the subculturing step; an assay step for assaying the amount of adenylate kinase 4 in the mesenchymal stem cells sampled in the sampling step; a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase 4; and a subculturing step for subculturing the sampled mesenchymal stem cells if the assayed amount of adenylate kinase 4 is equal to or higher than a predetermined threshold in the determination step; and these steps are repeated one or more times.
 9. The method for culturing mesenchymal stem cells according to claim 5, characterized in that the subculturing step comprises dividing the mesenchymal stem cells into plural groups and subculturing the plural groups.
 10. A method for producing a therapeutic agent for liver dysfunction, characterized by comprising: a collection step for collecting a bone marrow aspirate; an isolation step for isolating a fluid containing mesenchymal stem cells from the collected bone marrow aspirate; a culturing step for culturing mesenchymal stem cells in the isolated fluid; a sampling step for sampling cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity level of the mesenchymal stem cells from the assayed amount of adenylate kinase
 4. 11. A therapeutic agent for liver dysfunction, characterized in that the agent is produced by steps comprising: an isolation step for isolating a fluid; a culturing step for culturing mesenchymal stem cells in the isolated fluid; a sampling step for sampling cultured mesenchymal stem cells; an assay step for assaying the amount of adenylate kinase 4 in the sampled mesenchymal stem cells; and a determination step for determining the activity of the mesenchymal stem cells from the assayed amount of adenylate kinase
 4. 